There is substantial evidence that products of phosphatidylinositol 4,5-bisphosphate (PIP2), diacylglycerol (DG) and myo-inositol trisphosphate (IP3), can function as intracellular second messengers in a large variety of tissues. DG can activate protein kinase C (PKC) whereas IP3 mobilizes Ca2+ from ER. Previously, we have demonstrated that activation of alpha 1-adrenergic and 5-HT2 receptors in rabbit and bovine corneal epithelium provokes increased hydrolysis of PIP2 into DG and IP3 and that a GTP-binding protein is involved in coupling these receptors to phospholipase C (PLC). Corneal epithelium possesses active C1' transport system, undergoes mitosis and cell proliferation during wound healing which is facilitated by growth factors, and it is innervated by SP containing nerve fibers probably involved in nociception. The overall objective of the proposed work is to investigate if PIP2 and its metabolites have any role in corneal membrane functions. There are five specific aims of the proposed research plan: (1) We will investigate the effects of SP, EGF, adrenergic, serotonergic and cholinergic agonists on phosphoinositide metabolism in corneal epithelium. Various isomers of inositol phosphates will be identified by HPLC. (2) We will study the effect of IP3 on the release of CA2+ from corneal microsomal fraction and permeabilized epithelial cells. (3) We will study the subcellular distribution and biochemical characteristics of PLC involved in hydrolysis of phosphoinositides in corneal epithelium, stroma and endothelium. The effects of agents which alter the activity of fluid transport in the corneal endothelium on phosphoinositides will be investigated. (4) We will investigate the role and mechanism of GTP-binding protein in the coupling of agonist-receptors to PLC. (5) We will characterize PI-, PIP-and DG-kinase activities in the corneal epithelium, stroma and endothelium. We will investigate if there is a relationship between activation of PKC and DG-kinase activity in the corneal epithelium. The effects of polyamines on enzymes of phosphoinositide metabolism will also be investigated. It is hoped that the proposed studies will prove useful to obtain new and important information regarding the role of phosphoinositides and their derived second messengers in corneal membrane functions including C1' ion transport, epithelial cell migration and regeneration during wound healing, and nociception.